Emerging evidence supports a central role for chromatin insulators in genome-wide organization of higher- order chromosome loop structures. The overall objective of this proposal is to examine the consequences of insulator-mediated chromatin organization during mouse embryonic stem (ES) cell differentiation and development. Our central hypothesis is that the vertebrate insulator CTCF partitions the genome into lineage-specific domains of co-expressed genes by facilitating the formation of higher-order chromatin loop structures in response to developmental cues. This hypothesis will be tested according to three specific aims. In Aim 1, live cell confocal microscopy will be leveraged to investigate CTCF distribution and dynamics in real time during ES cell commitment along the neural lineage. In Aim 2, genome-wide CTCF binding sites will be identified in ES cells using chromatin immunoprecipitation in combination with high- throughput, next generation sequencing (ChlP-Seq). Global CTCF occupancy maps for pluripotent ES cells will be compared to multipotent neuroprogenitors and terminally-differentiated neurons. Finally, in Aim 3, the structural organization of CTCF-based chromatin loops will be characterized using Chromosome- Conformation-Capture (3C) at genomic loci displaying differential insulator occupancy during neural lineage commitment. Correlation of these CTCF-based structures with recent genome-wide analyses of 'traditional'epigenetic modifications and lineage-specific gene expression profiles will provide a more global understanding of how the genome and the epigenome act in concert to regulate the formation of a diverse array of tissue-types during development. Completion of the proposed work will provide significant insight into the mechanisms that govern the commitment of pluripotent stem cells toward neuroectodermal lineages. This knowledge will enable advances in understanding the causes and consequences of higher- order chromatin structure during the onset of cancer and neurodegenerative diseases. Public Health Relevance: Embryonic stem cells have enormous potential for regenerative medicine due to their capacity for indefinite self-renewal while remaining poised for differentiation into all adult cell-types. The proposed research will enhance our understanding of the molecular mechanisms that regulate commitment of stem cells along the neural lineage during embryonic development. This knowledge will provide a foundation for the development of robust strategies which harness the therapeutic potential of stem cells for treatment of neurodegenerative diseases and brain cancer.